McDonagh Brian, Ogueta Samuel, Lasarte Guillermo, Padilla C Alicia, Bárcena José Antonio
Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Universidad de Córdoba, Cordoba, Spain.
J Proteomics. 2009 May 2;72(4):677-89. doi: 10.1016/j.jprot.2009.01.023.
Post-translational redox modification of thiol groups can form the molecular basis of antioxidative protection and redox control. We have implemented a shotgun redox proteomic technique to identify the precise cysteines reversibly oxidised in key proteins. The method was applied to Saccharomyces cerevisiae subjected to peroxide treatment. Enrichment by covalent redox affinity chromatography allowed the isolation of a "redox subpeptidome" that was analysed by LC-MS/MS. Unique peptides containing specific reversibly oxidised cysteines were used to identify over 70 proteins in control and treated samples of which 27 were consistently present in all replicates. In most cases, the redox modification negatively affects their function and slows down their metabolic pathways. Integration of the data provides a snapshot consistent with a metabolic defensive strategy, regulating key enzymes by redox modification, redirecting energy toward ribulose-5-phosphate recycling for NADPH production and antioxidative defence.This generally applicable method has allowed us to discover new redox regulated proteins (DAHP and carbamoylphosphate synthases, Doa1p) and to precisely identify target cysteines in a number of known ones.
硫醇基团的翻译后氧化还原修饰可构成抗氧化保护和氧化还原控制的分子基础。我们采用了鸟枪法氧化还原蛋白质组学技术来鉴定关键蛋白质中可逆氧化的精确半胱氨酸。该方法应用于经过过氧化物处理的酿酒酵母。通过共价氧化还原亲和色谱进行富集，得以分离出一个“氧化还原亚肽组”，并通过液相色谱-串联质谱进行分析。含有特定可逆氧化半胱氨酸的独特肽段用于鉴定对照样品和处理样品中的70多种蛋白质，其中27种在所有重复实验中均始终存在。在大多数情况下，氧化还原修饰会对其功能产生负面影响，并减缓其代谢途径。数据整合提供了一个与代谢防御策略一致的概况，即通过氧化还原修饰调节关键酶，将能量重新导向5-磷酸核酮糖循环以产生NADPH并进行抗氧化防御。这种普遍适用的方法使我们能够发现新的氧化还原调节蛋白（DAHP和氨甲酰磷酸合酶、Doa1p），并精确鉴定许多已知蛋白中的目标半胱氨酸。
